The present invention relates to a delivery system. In particular, the present invention relates to a retroviral vector capable of delivering a nucleotide sequence of interest (hereinafter abbreviated to xe2x80x9cNOIxe2x80x9d)xe2x80x94or even a plurality of NOIsxe2x80x94to a site of interest.
More in particular, the present invention relates to a retroviral vector useful in gene therapy.
Gene therapy includes any one or more of: the addition, the replacement, the deletion, the supplementation, the manipulation etc. of one or more nucleotide sequences in, for example, one or more targetted sitesxe2x80x94such as targetted cells. If the targetted sites are targetted cells, then the cells may be part of a tissue or an organ. General teachings on gene therapy may be found in Molecular Biology (Ed Robert Meyers, Pub VCH, such as pages 556-558).
By way of further example, gene therapy also provides a means by which any one or more of: a nucleotide sequence, such as a gene, can be applied to replace or supplement a defective gene; a pathogenic gene or gene product can be eliminated; a new gene can be added in order, for example, to create a more favourable phenotype; cells can be manipulated at the molecular level to treat cancer (Schmidt-Wolf and Schmidt-Wolf, 1994, Annals of Hematology 69;273-279) or other conditionsxe2x80x94such as immune, cardiovascular, neurological, inflammatory or infectious disorders; antigens can be manipulated and/or introduced to elicit an immune responsexe2x80x94such as genetic vaccination.
In recent years, retroviruses have been proposed for use in gene therapy. Essentially, retroviruses are RNA viruses with a life cycle different to that of lytic viruses. In this regard, when a retrovirus infects a cell, its genome is converted to a DNA form. In otherwords, a retrovirus is an infectious entity that replicates through a DNA intermediate. More details on retroviral infection etc. are presented later on.
There are many retroviruses and examples include: murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinarni sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).
A detailed list of retroviruses may be found in Coffin et al (xe2x80x9cRetrovirusesxe2x80x9d 1997 Cold Spring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp 758-763).
Details on the genomic structure of some retroviruses may be found in the art. By way of example, details on HIV may be found from the NCBI Genbank (i.e. Genome Accession No. AF033819).
All retroviruses contain three major coding domains, gag, pol, env, which code for essential virion proteins. Nevertheless, retroviruses may be broadly divided into two categories: namely, xe2x80x9csimplexe2x80x9d and xe2x80x9ccomplexxe2x80x9d. These categories are distinguishable by the organisation of their genomes. Simple retroviruses usually carry only this elementary information. In contrast, complex retroviruses also code for additional regulatory proteins derived from multiple spliced messages.
Retroviruses may even be further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses. A review of these retroviruses is presented in xe2x80x9cRetrovirusesxe2x80x9d (1997 Cold Spring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp 1-25).
All oncogenic members except the human T-cell leukemia virus-bovine leukemia virus (HTLV-BLV) are simple retroviruses. HTLV, BLV and the lentiviruses and spumaviruses are complex. Some of the best studied oncogenic retroviruses are Rous sarcoma virus (RSV), mouse mammary tumour virus (MMTV) and murine leukemia virus (MLV) and the human T-cell leukemia virus (HTLV).
The lentivirus group can be split even further into xe2x80x9cprimatexe2x80x9d and xe2x80x9cnon-primatexe2x80x9d. Examples of primate lentiviruses include the human immunodeficiency virus (HIV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV). The non-primate lentiviral group includes the prototype xe2x80x9cslow virusxe2x80x9d visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiencey virus (FIV) and bovine immunodeficiencey virus (BIV).
A critical distinction between the lentivirus family and other types of retroviruses is that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis et al 1992 EMBO. J 11; 3053-3058, Lewis and Emerman 1994 1. Virol. 68: 510-516). In contrast, other retrovirusesxe2x80x94such as MLVxe2x80x94are unable to infect non-dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.
During the process of infection, a retrovirus initially attaches to a specific cell surface receptor. On entry into the susceptible host cell, the retoviral RNA genome is then copied to DNA by the virally encoded reverse transcriptase which is carried inside the parent virus. This DNA is transported to the host cell nucleus where it subsequently integrates into the host genome. At this stage, it is typically referred to as the provirus. The provirus is stable in the host chromosome during cell division and is transcribed like other cellular proteins. The provirus encodes the proteins and packaging machinery required to make more virus, which can leave the cell by a process sometimes called xe2x80x9cbuddingxe2x80x9d.
As already indicated, each retroviral genome comprises genes called gag, pol and env which code for virion proteins and enzymes. These genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration, and transcription. They also serve as enhancer-promoter sequences. In other words, the LTRs can control the expression of the viral gene. Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5xe2x80x2 end of the viral genome.
The LTRs themselves are indentical sequences that can be divided into three elements, which are called U3, R and U5. U3 is derived from the sequence unique to the 3xe2x80x2 end of the RNA. R is derived from a sequence repeated at both ends of the RNA and U5 is derived from the sequence unique to the 5xe2x80x2 end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.
For ease of understanding, a simple, generic diagram (not to scale) of a retroviral genome showing the elementary features of the LTRs, gag, pol and env is presented in FIG. 6.
For the viral genome, the site of transcription initiation is at the boundary between U3 and R in the left hand side LTR (as shown in FIG. 6) and the site of poly (A) addition (termination) is at the boundary between R and US in the right hand side LTR (as shown in FIG. 6). U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins. Some retroviruses have any one or more of the following genes that code for proteins that are involved in the regulation of gene expression: tat, rev, tax and rex.
With regard to the structural genes gag, pol and env themselves, gag encodes the internal structural protein of the virus. Gag is proteolytically processed into the mature proteins MA (matrix), CA (capsid), NC (nucleocapsid). The gene pol encodes the reverse transcriptase (RT), which contains both DNA polymerase, and associated RNase H activities and integrase (IN), which mediates replication of the genome. The gene env encodes the surface (SU) glycoprotein and the transmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. This interaction leads ultimately to fusion of the viral membrane with the cell membrane.
The envelope glycoprotein complex of retroviruses includes two polypeptides: an external, glycosylated hydrophilic polypeptide (SU) and a membrane-spanning protein (TM). Together, these form an oligomeric xe2x80x9cknobxe2x80x9d or xe2x80x9cknobbed spikexe2x80x9d on the surface of a virion. Both polypeptides are encoded by the env gene and are synthesised in the form of a polyprotein precursor that is proteolytically cleaved during its transport to the cell surface. Although uncleaved Env proteins are able to bind to the receptor, the cleavage event itself is necessary to activate the fusion potential of the protein, which is necessary for entry of the virus into the host cell. Typically, both SU and TM proteins are glycosylated at multiple sites. However, in some viruses, exemplified by MLV, TM is not glycosylated.
Although the SU and TM proteins are not always required for the assembly of enveloped virion particles as such, they do play an essential role in the entry process. In this regard, the SU domain binds to a receptor moleculexe2x80x94often a specific receptor moleculexe2x80x94on the target cell. It is believed that this binding event activates the membrane fusion-inducing potential of the TM protein after which the viral and cell membranes fuse. In some viruses, notably MLV, a cleavage eventxe2x80x94resulting in the removal of a short portion of the cytoplasmic tail of TMxe2x80x94is thought to play a key role in uncovering the full fusion activity of the protein (Brody et al 1994 J. Virol. 68: 4620-4627, Rein et al 1994 J. Virol. 68: 1773-1781). This cytoplasmic xe2x80x9ctailxe2x80x9d, distal to the membrane-spanning segment of TM remains on the internal side of the viral membrane and it varies considerably in length in different retroviruses.
Thus, the specificity of the SU/receptor interaction can define the host range and tissue tropism of a retrovirus. In some cases, this specificity may restrict the transduction potential of a recombinant retroviral vector. For this reason, many gene therapy experiments have used MLV. A particular MLV that has an envelope protein called 4070A is known as an amphotropic virus, and this can also infect human cells because its envelope protein xe2x80x9cdocksxe2x80x9d with a phosphate transport protein that is conserved between man and mouse. This transporter is ubiquitous and so these viruses are capable of infecting many cell types. In some cases however, it may be beneficial, especially from a safety point of view, to specifically target restricted cells. To this end, several groups have engineered a mouse ecotropic retrovirus, which unlike its amphotropic relative normally only infects mouse cells, to specifically infect particular human cells. Replacement of a fragment of an envelope protein with an erythropoietin segement produced a recombinant retrovirus which then bound specifically to human cells that expressed the erythropoietin receptor on their surface, such as red blood cell precursors (Maulik and Patel 1997 xe2x80x9cMolecular Biotechnology: Therapeutic Applications and Strategiesxe2x80x9d 1997. Wiley-Liss linc. pp 45.).
In addition to gag, pol and env, the complex retroviruses also contain xe2x80x9caccessoryxe2x80x9d genes which code for accessory or auxiliary proteins. Accessory or auxiliary proteins are defined as those proteins encoded by the accessory genes in addition to those encoded by the usual replicative or structural genes, gag, pol and env. These accessory proteins are distinct from those involved in the regulation of gene expression, like those encoded by rat, rev, tax and rex. Examples of accessory genes include one or more of vif, vpr, vpx, vpu and nef. These accessory genes can be found in, for example, HIV (see, for example pages 802 and 803 of xe2x80x9cRetrovirusesxe2x80x9d Ed. Coffin et al Pub. CSHL 1997). Non-essential accessory proteins may function in specialised cell types, providing functions that are at least in part duplicative of a function provided by a cellular protein. Typically, the accessory genes are located between pol and env, just downstream from env including the U3 region of the LTR or overlapping portions of the env and each other.
The complex retroviruses have evolved regulatory mechanisms that employ virally encoded transcriptional activators as well as cellular transcriptional factors. These trans-acting viral proteins serve as activators of RNA transcription directed by the LTRs. The transcriptional trans-activators of the lentiviruses are encoded by the viral tat genes. Tat binds to a stable, stem-loop, RNA secondary structure, referred to as TAR, one function of which is to apparently optimally position Tat to trans-activate transcription.
As mentioned earlier, retroviruses have been proposed as a delivery system (other wise expressed as a delivery vehicle or delivery vector) for inter alia the transfer of a NOI, or a plurality of NOIs, to one or more sites of interest. The transfer can occur in vitro, ex vivo, in vivo, or combinations thereof. When used in this fashion, the retroviruses are typically called retroviral vectors or recombinant retroviral vectors. Retroviral vectors have even been exploited to study various aspects of the retrovirus life cycle, including receptor usage, reverse transcription and RNA packaging (reviewed by Miller, 1992 Curr Top Microbiol Immunol 158:1-24).
In a typical recombinant retroviral vector for use in gene therapy, at least part of one or more of the gag, pol and env protein coding regions may be removed from the virus. This makes the retroviral vector replication-defective. The removed portions may even be replaced by a NOI in order to generate a virus capable of integrating its genome into a host genome but wherein the modified viral genome is unable to propagate itself due to a lack of structural proteins. When integrated in the host genome, expression of the NOI occursxe2x80x94resulting in, for example, a therapeutic effect. Thus, the transfer of a NOI into a site of interest is typically achieved by: integrating the NOI into the recombinant viral vector; packaging the modified viral vector into a virion coat; and allowing transduction of a site of interestxe2x80x94such as a targetted cell or a targetted cell population.
It is possible to propagate and isolate quantities of retroviral vectors (e.g. to prepare suitable titres of the retroviral vector) for subsequent transduction of, for example, a site of interest by using a combination of a packaging or helper cell line and a recombinant vector.
In some instances, propagation and isolation may entail isolation of the retroviral gag, pol and env genes and their separate introduction into a host cell to produce a xe2x80x9cpackaging cell linexe2x80x9d. The packaging cell line produces the proteins required for packaging retroviral DNA but it cannot bring about encapsidation due to the lack of a psi region. However, when a recombinant vector carrying a NOI and a psi region is introduced into the packaging cell line, the helper proteins can package the psi-positive recombinant vector to produce the recombinant virus stock. This can be used to infect cells to introduce the NOI into the genome of the cells. The recombinant virus whose genome lacks all genes required to make viral proteins can infect only once and cannot propagate. Hence, the NOI is introduced into the host cell genome without the generation of potentially harmful retrovirus. A summary of the available packaging lines is presented in xe2x80x9cRetrovirusesxe2x80x9d (1997 Cold Spring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp 449).
However, this technique can be problematic in the sense that the titre levels are not always at a satisfactory level. Nevertheless, the design of retroviral packaging cell lines has evolved to address the problem of inter alia the spontaneous production of helper virus that was frequently encountered with early designs. As recombination is greatly facilitated by homology, reducing or eliminating homology between the genomes of the vector and the helper has reduced the problem of helper virus production.
More recently, packaging cells have been developed in which the gag, pol and env viral coding regions are carried on separate expression plasmids that are independently transfected into a packaging cell line so that three recombinant events are required for wild type viral production. This strategy is sometimes referred to as the three plasmid transfection method (Soneoka et al 1995 Nucl. Acids Res. 23: 628-633).
Transient transfection can also be used to measure vector production when vectors are being developed. In this regard, transient transfection avoids the longer time required to generate stable vector-producing cell lines and is used if the vector or retroviral packaging components are toxic to cells. Components typically used to generate retroviral vectors include a plasmid encoding the Gag/Pol proteins, a plasmid encoding the Env protein and a plasmid containing, a NOI. Vector production involves transient transfection of one or more of these components into cells containing the other required components. If the vector encodes toxic genes or genes that interfere with the replication of the host cell, such as inhibitors of the cell cycle or genes that induce apotosis, it may be difficult to generate stable vector-producing cell lines, but transient transfection can be used to produce the vector before the cells die. Also, cell lines have been developed using transient infection that produce vector titre levels that are comparable to the levels obtained from stable vector-producing cell lines (Pear et al 1993, PNAS 90:8392-8396).
In view of the toxicity of some HIV proteinsxe2x80x94which can make it difficult to generate stable HIV-based packaging cellsxe2x80x94HIV vectors are usually made by transient transfection of vector and helper virus. Some workers have even replaced the HIV Env protein with that of vesicular stomatis virus (VSV). Insertion of the Env protein of VSV facilitates vector concentration as HIVIVSV-G vectors with titres of 5xc3x97105 (108 after concentration) were generated by transient transfection (Naldini et al 1996 Science 272: 263-267). Thus, transient transfection of HIV vectors may provide a useful strategy for the generation of high titre vectors (Yee et al 1994 PNAS. 91: 9564-9568). A drawback, however, with this approach is that the VSV-G protein is quite toxic to cells.
Replacement of the env gene with a heterologous ,env gene is an example of a technique or strategy called pseudotyping. Pseudotyping is not a new phenomenon and examples may be found in WO-A-98/05759, WO-A-98/05754, WO-A-97/17457, WO-A-96/09400, WO-A-91/00047 and Mebatsion et al 1997 Cell 90, 841-847.
Pseudotyping can confer one or more advantages. For example, with the lentiviral vectors, the env gene product of the HIV based vectors would restrict these vectors to infecting only cells that express a protein called CD4. But if the env gene in these vectors has been substituted with env sequences from other RNA viruses, then they may have a broader infectious spectrum (Verma and Somia 1997 Nature 389:239-242). As just describedxe2x80x94and by way of examplexe2x80x94workers have pseudotyped an HIV based vector with the glycoprotein from VSV (Verina and Somia 1997 ibid).
Also, and by way of example, the relative fragility of the retroviral Env protein may limit the ability to concentrate retroviral vectorsxe2x80x94and concentrating the virus may result in a poor viral recovery. To some extent, this problem may be overcome by substitution of the retroviral Env protein with the more stable VSV-G protein allowing more efficient vector concentration with better yields (Naldini et al 1996. Science 272: 263-267).
However, pseudotyping with VSV-G protein may not always be desirable. This is because the VSV-G protein is cytotoxic (Chen et al 1996, Proc. Natl. Acad. Sci. 10057 and references cited therein).
Hence, it is desirable to find other proteins which are non-toxic and which can be used to pseudotype a retroviral vector.
Thus, the present invention seeks to provide an improved retroviral vector.
According to a first aspect of the present invention there is provided a retroviral delivery system capable of transducing a target site, wherein the retroviral delivery system comprises a first nucleotide sequence coding for at least a part of an envelope protein; and one or more other nucleotide sequences derivable from a retrovirus that ensure transduction of the target site by the retroviral delivery system; wherein the first nucleotide sequence is heterologous with respect to at least one of the other nucleotide sequences; and wherein the first nucleotide sequence codes for at least a part of a rabies G protein or a mutant, variant, derivative or fragment thereof that is capable of recognising the target site.
The retroviral delivery system of the present invention can comprise one entity. Alternatively, the retroviral delivery system of the present invention can comprise a plurality of entities which in combination provide the retroviral delivery system of the present invention. Examples of these viral delivery systems can include but are not limited to herpesviruses and adenoviruses as described in Savard et al 1997, J Virol 71(5): 4111-4117; Feng et al 1997, Nat Biotechnol 15(9): 866-870; and UK Patent Application No. 9720465.5.
The term xe2x80x9cderivablexe2x80x9d is used in its normal sense as meaning the sequence need not necessarily be obtained from a retrovirus but instead could be derived therefrom. By way of example, the sequence may be prepared synthetically or by use of recombinant DNA techniques.
According to a second aspect of the present invention there is provided a viral particle obtainable from the retroviral delivery system according to the present invention.
According to a third aspect of the present invention there is provided a retroviral vector wherein the retroviral vector is the retroviral delivery system according to the first aspect of the present invention or is obtainable therefrom.
According to a fourth aspect of the present invention there is provided a cell transduced with a retroviral delivery system according to the present invention, or a viral particle according to the present invention, or a retroviral vector according to the present invention.
According to a fifth aspect of the present invention there is provided a retoviral delivery system according to the present invention, or a viral particle according to the present invention, or a retroviral vector according to the present invention, for use in medicine.
According to a sixth aspect of the present invention there is provided the use of a retroviral delivery system according to the present invention, or a viral particle according to the present invention, or a retroviral vector according to the present invention in the manufacture of a pharmaceutical composition to deliver a NOI to a target site in need of same.
According to a seventh aspect of the present invention there is provided a method comprising contacting a cell with a retroviral delivery system according to the present invention, or a viral particle according to the present invention, or a retroviral vector according to the present invention.
According to an eighth aspect of the present invention there is provided a vector for preparing a retroviral delivery system according to the present invention, or a viral particle according to the present invention, or a retroviral vector according to the present invention, wherein the vector comprises a nucleotide sequence coding for at least a part of the rabies G protein or a mutant, variant, derivative or fragment thereof.
According to a ninth aspect of the present invention there is provided a plasmid for preparing a retroviral delivery system according to the present invention, or a viral particle according to the present invention, or a retroviral vector according to the present invention, wherein the plasmid comprises a nucleotide sequence coding for at least a part of the rabies G protein or a mutant, variant, derivative or fragment thereof.
According to a tenth aspect of the present invention there is provided a plurality of plasmids, wherein at least one plasmid is a plasmid according to the present invention and wherein at least one other plasmid comprises one or more nucleotide sequences derivable from a retrovirus.
According to an eleventh aspect of the present invention there is provided the use of a rabies G protein to pseudotype a retrovirus or a retroviral vector or a retroviral particle in order to affect the infectious profile of the retrovirus or the retroviral vector or the retroviral particle.
According to a eleventh aspect of the present invention there is provided the use of a rabies G protein to pseudotype a retrovirus or a retroviral vector or a retroviral particle in order to affect the host range and/or cell tropism of the retrovirus or the retroviral vector or the retroviral particle.
According to a thirteenth aspect of the present invention there is provided a retrovirus or a retroviral vector or a retroviral particle pseudotyped with a rabies G protein.
According to a fourteenth aspect of the present invention there is provided a retroviral delivery system comprising a heterologous env region, wherein the heterologous env region comprises at least a part of a nucleotide sequence coding for a rabies G protein.
According to a fifteenth aspect of the present invention there is provided a retroviral delivery system comprising a heterologous env region, wherein the heterologous env region comprises a nucleotide sequence coding for a rabies G protein.
Preferably the first nucleotide sequence codes for all of a rabies G protein or a mutant, variant, derivative or fragment thereof.
Preferably at least one of the other nucleotide sequences is derivable from a lentivirus or an oncoretrovirus.
Preferably the other nucleotide sequences are derivable from a lentivirus or an oncoretrovirus.
Preferably the other nucleotide sequences are derivable from MLV, HIV or EIAV.
Preferably the retroviral delivery system comprises at least one NOI.
Preferably the NOI has a therapeutic effect or codes for a protein that has a therapeutic effect.
Preferably the target site is a cell.
Thus the present invention provides a retroviral vector having a heterologous envelope protein. This retroviral vector is useful in gene therapy.
An important aspect of the present invention is the pseudotyping of a retrovirus, and/or a retroviral vector derivable or based on same, with a nucleotide sequence coding for a rabies protein, especially the rabies G protein. Here, the term pseudotyping means incorporating in at least a part of, or substituting a part of, or replacing all of, an env gene of a viral genome, or of a viral vector, a protein from another virus.
Teachings on the rabies G protein, as well as mutants thereof, may be found in Rose et al., 1982 J. Virol. 43: 361-364, Hanham et al., 1993 J. Virol.,67, 530-542, Tuffereau et al.,1998 J. Virol., 72, 1085-1091, Kucera et al., 1985 J. Virol 55, 158-162, Dietzschold et al., 1983 PNAS 80, 70-74, Seif et al., 1985 J. Virol., 53, 926-934, Coulon et al.,1998 J. Virol., 72, 273-278, Tuffereau et al.,1998 J. Virol., 72, 1085-10910, Burger et al., 1991 J. Gen. Virol. 72. 359-367, Gaudin et al 1995 J Virol 69, 5528-5534, Bennansour et al 1991 J Virol 65, 41984203, Luo et al 1998 Microbiol Immunol 42, 187-193, Coll 1997 Arch Virol 142, 2089-2097, Luo et al 1997 Virus Res 51, 35-41, Luo et al 1998 Microbiol Immunol 42, 187-193, Coll 1995 Arch Virol 140, 827-851, Tuchiya et al 1992 Virus Res 25, 1-13, Morirnoto et al 1992 Virology 189, 203-216, Gaudin et al 1992 Virology 187, 627-632, Whitt et al 1991 Virology 185, 681-688, Dietzschold et al 1978 J Gen Virol 40, 131-139, Dietzschold et al 1978 Dev Biol Stand 40, 45-55, Dietzschold et at 1977 I Virol 23, 286-293, and Otvos et al 1994 Biochim Biophys Acta 1224, 68-76. A rabies G protein is also described in EP-A-0445625.
The use of rabies G protein according to the invention provides vectors which in vivo preferentially transduce targetted cells which rabies virus preferentially infects. This includes in particular neuronal target cells in vivo. For a neuron-targeted vector, rabies G from a pathogenic strain of rabies such as ERA may be particularly effective. On the other hand rabies G protein confers a wider target cell range in vitro including nearly all mammalian and avian cell types tested (Seganti el at., 1990 Arch Virol. 34,155-163; Fields et al., 1996 Fields Virology, Third Edition, vol.2, Lippincott-Raven Publishers, Philadelphia, N.Y.). It is likely that it will be found also to confer an ability to infect other cell types which will be of interest. Thus, the use of rabies G protein according to the invention also enables the provision of vectors which transduce a wide variety of cell types in vitro and also in vivo. The different tropism of rabies virus in vivo and in vitro is thought to be due to the ability of rabies G to bind to a series of receptors, some of which are only active in vitro.
Alternatively, the tropism of the pseudotyped vector particles according to the invention may be modified by the use of a mutant rabies G which is modified in the extracellular domain. Rabies G protein has the advantage of being mutatable to restrict target cell range. The uptake of rabies virus by target cells in vivo is thought to be mediated by the acetylcholine receptor (AchR) but there may be other receptors to which in binds in vivo (Hanham el al., 1993 J. Virol.,67, 530-542; Tuffereau et al.,1998 J. Virol., 72, 1085-1091). The effects of mutations in antigenic site III of the rabies G protein on virus tropism have been investigated, this region is not thought to be involved in the binding of the virus to the acetylcholine receptor (Kucera et al., 1985 J. Virol 55, 158-162; Dietzschold et al., 1983 Proc Natl Acad Sci 80, 70-74; Seif er al., 1985 J.Virol., 53, 926-934; Coulon et al.,1998 J. Virol., 72, 273-278; Tuffereau et al.,1998 J. Virol., 72, 1085-10910). For example a mutation of the arginine at amino acid 333 in the mature protein to glutamine can be used to restrict viral entry to olfactory and peripheral neurons in vivo while reducing propagation to the central nervous system. These viruses were able to penetrate motorneurons and sensory neurons as efficiently as the wt virus, yet transneuronal transfer did not occur (Coulon et al., 1989, J. Virol. 63, 3550-3554). Viruses in which amino acid 330 has been mutated are further attenuated, being unable to to infect either motorneurons or sensory neurons after intramuscular injection (Coulon et al.,1998 J. Virol., 72, 273-278).
Alternatively or additionally, rabies G proteins from laboratory passaged strains of rabies may be used. These can be screened for alterations in tropism. Such strains include the following:
By way of example, the ERA strain is a pathogenic strain of rabies and the rabies G protein from this strain can be used for transduction of neuronal cells. The sequence of rabies G from the ERA strains is in the GenBank database (accession number J02293). This protein has a signal peptide of 19 amino acids and the mature protein begins at the lysine residue 20 amino acids from the translation initiation methionine. The HEP-Flury strain contains the mutation from arginine to glutamine at amino acid position 333 in the mature protein which correlates with reduced pathogenicity and which can be used to restrict the tropism of the viral envelope.
An example of a rabies G protein is shown as SEQ ID No. 2 and its coding sequence is presented as SEQ ID No. 1. The present invention covers variants, homologues or derivatives of those sequences.
The terms xe2x80x9cvariantxe2x80x9d, xe2x80x9chomologuexe2x80x9d or xe2x80x9cfragmentxe2x80x9d in relation to the amino acid sequence for the preferred enzyme of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence providing the resultant protein has G protein activity and/or G protein characteristics or profile, preferably being at least as biologically active as the G protein shown as SEQ ID No. 2. In particular, the term xe2x80x9chomologuexe2x80x9d covers homology with respect to structure and/or function. With respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequence shown as SEQ ID No. 2. More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown as SEQ ID No. 2.
The terms xe2x80x9cvariantxe2x80x9d, xe2x80x9chomologuexe2x80x9d or xe2x80x9cfragmentxe2x80x9d in relation to the nucleotide sequence coding for the preferred enzyme of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for or is capable of coding for a protein having G protein activity and/or G protein characteristics or profile, preferably being at least as biologically active as the G protein encoded by the sequences shown as SEQ ID No. 1. In particular, the term xe2x80x9chomologuexe2x80x9d covers homology with respect to structure and/or function providing the resultant nucleotide sequence codes for or is capable of coding for a protein having G protein activity and/or G protein characteristics or profile. With respect to sequence homology, preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequence shown as SEQ ID No. 1. More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown as SEQ ID No. 1.
In particular, the term xe2x80x9chomologyxe2x80x9d as used herein may be equated with the term xe2x80x9cidentityxe2x80x9d. Relative sequence homology (i.e. sequence identity) can be determined by commercially available computer programs that can calculate % homology between two or more sequences. A typical example of such a computer program is CLUSTAL.
The terms xe2x80x9cvariantxe2x80x9d, xe2x80x9chomologuexe2x80x9d or xe2x80x9cfragmentxe2x80x9d are synonymous with allelic variations of the sequences.
The term xe2x80x9cvariantxe2x80x9d also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequence presented herein. Preferably, the term xe2x80x9cvariantxe2x80x9d encompasses sequences that are complementary to sequences that are capable of hybridising under stringent conditions (e.g. 65xc2x0 C. and 0.1 SSC {1xc3x97 SSC=0.15 M NaCl, 0.015 Na3 citrate pH 7.0}) to the nucleotide sequence presented herein.
The present invention also covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention (including complementary sequences of those presented herein). In a preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention under stringent conditions (e.g. 65xc2x0 C. and 0.1 SSC) to the nucleotide sequence presented herein (including complementary sequences of those presented herein).
A major advantage of using the rabies glycoprotein in comparison to the VSV glycoprotein is the detailed knowledge of its toxicity to man and other animals due to the extensive use of rabies vaccines. In particular phase 1 clinical trials have been reported on the use of rabies glycoprotein expressed from a canarypox recombinant virus as a human vaccine (Fries et al., 1996 Vaccine 14, 428-434), these studies concluded that the vaccine was safe for use in humans.
The retroviral vectors of the present invention are useful for the delivery of one or more NOIs to cells in vivo and in vitro, in particular the delivery of therapeutically active NOI(s). One or more selected NOI(s) may be incorporated in the vector genome for expression in the target cell. The NOI(s) may have one or more expression control sequences of their own, or their expression may be controlled by the vector LTRs. For appropriate expression of the NOI(s), a promoter may be included in or between the LTRs which is preferentially active under certain conditions or in certain cell types. The NOI may be a sense sequence or an antisense sequence. Furthermore, if there is a plurality of NOIs then those NOIs may be sense sequences or antisense sequences or combinations thereof.
The retroviral vector genome of the present invention may generally comprise LTRs at the 5xe2x80x2 and 3xe2x80x2 ends, one or more NOI(s) including therapeutically active genes and/or marker genes, or suitable insertion sites for inserting one or more NOI(s), and a packaging signal to enable the genome to be packaged into a vector particle in a producer cell. There may even be suitable primer binding sites and integration sites to allow reverse transcription of the vector RNA to DNA, and integration of the proviral DNA into the target cell genome. In a preferred embodiment, the retroviral vector particle has a reverse transcription system (compatible reverse transcription and primer binding sites) and an integration system (compatible integrase and integration sites).
Thus, in accordance with the present invention, it is possible to manipulate the viral genome or the retroviral vector nucleotide sequence, so that viral genes are replaced or supplemented with one or more NOIs. The NOI(s) may be any one or more of selection gene(s), marker gene(s) and therapeutic gene(s). Many different selectable markers have been used successfully in retroviral vectors. These are reviewed in xe2x80x9cRetrovirusesxe2x80x9d (1997 Cold Spring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, HE Varmus pp 444) and include, but are not limited to, the bacterial neomycin and hygromycin phosphotransferase genes which confer resistance to G418 and hygromycin respectively; a mutant mouse dihydrofolate reductase gene which confers resistance to methotrexate; the bacterial gpt gene which allows cells to grow in medium containing mycophenolic acid, xanthine and aminopterin; the bacterial hisD gene which allows cells to grow in medium without histidine but containing histidinol; the multidrug resistance gene (mdr) which confers resistance to a variety of drugs; and the bacterial genes which confer resistance to puromycin or phleomycin. All of these markers are dominant selectable and allow chemical selection of most cells expressing these genes.
In accordance with the present invention, the NOI can be a therapeutic genexe2x80x94in the sense that the gene itself may be capable of eliciting a therapeutic effect or it may code for a product that is capable of eliciting a therapeutic effect.
Non-limiting examples of therapeutic NOIs include genes encoding tumour supressor proteins, cytokines, anti-viral proteins, immunomodulatory molecules, antibodies, engineered immunoglobulin-like molecules, fusion proteins, hormones, membrane proteins, vasoactive proteins or peptides, growth factors, ribozymes, antisense RNA, enzymes, prorugs, such as pro-drug activating enzymes, cytotoxic agents, and enzyme inhibitors.
Examples of prodrugs include but are not limited to etoposide phosphate (used with alkaline phosphatase; 5-fluorocytosine (with cytosine deaminase); Doxorubin-N-p-hydroxyphenoxyacetarnide (with Penicillin-V-Amidase); Para-N-bis (2-chloroethyl)aminobenzoyl glutamate (with Carboxypeptidase G2); Cephalosporin nitrogen mustard carbamates (with B-lactamase); SR4233 (with p450 reductase); Ganciclovir (with HSV thyridine kinase); mustard pro-drugs with nitroreductase and cyclophosphamide or ifosfamide (with cytochrome p450).
Diseases which may be treated include, but are not limited to cancer, heart disease, stroke, neurodegenerative disease, arthritis, viral infection, bacterial infection, parasitic infection, diseases of the imrnune system, viral infection, tumours, blood clotting disorders, and genetic diseasesxe2x80x94such as chronic granulomatosis, Lesch-Nyhan sysndrome, Parkinson""s disease, empysema, phenylketonuria, sickle cell anaemia,xcex1-thalasemia, xcex2-thalasemia, Gaucher disease.
Target cells for gene therapy using retroviral vectors include but are not limited to haematopoietic cells, (including monocytes, macrophages, lymphocytes, granulocytes, or progenitor cells of any of these); endothelial cells, tumour cells, stromal cells, astrocytes, or glial cells, muscle cells, epithelial cells, neurons, fibroblasts, hepatocyte. astrocyte, and lung cells.
Within the retroviral vector of the present invention, the one or more NOIs can be under the transcriptional control of the viral LTRs. Alternatively, a combination of enhancer-promotcr elements can be present in order to achieve higher levels of expression. The promoter-enhancer elements are preferably strongly active or capable of being strongly induced in the target cells. An example of a strongly active promoter-enhancer combination is a human cytomegalovirus (HCMV) major intermediate early (MIE) promoter/enhancer combination. The promoter-enhancer combination may be tissue or temporally restricted in their activity. Examples of a suitable tissue restricted promoter-enhancer combinations are those which are highly active in tumour cells such as a promoter-enhancer combination from a MUCI gene or a CEA gene.
Hypoxia or ischaemia regulatable expression may also be particularly useful under certain circumstances. Hypoxia is a powerful regulator of gene expression in a wide range of different cell types and acts by the induction of the activity of hypoxia-inducible transcription factors such as hypoxia inducible factor-1 (HIF-1) (Wang and Semenza 1993 PNAS. (USA) 90: 430) which bind to cognate DNA recognition sites, the hypoxia responsive elements (HREs) on various gene promoters. A multimeric form of HRE from the mouse phosphoglycerate kinase-1 (PGK-1) gene has been used to control expression of both marker and therapeutic genes by human fibrosarcoma cells in response to hypoxia in vitro and within solid tumours in vivo (Firth et al 1994, PNAS 91(14): 6496-6500; Dachs et al 1997 Nature Med. 5: 515). Alternatively, the fact that glucose deprivation is also present in ischaemic areas of tumours can be used to activate heterologous gene expression especially in tumours. A truncated 632 base pair sequence of the grp 78 gene promoter, known to be activated specifically by glucose deprivation, has been shown to be capable of driving high level expression of a reporter gene in murine rumours in vivo (Gazit et al 1995 Cancer Res. 55: 1660.).
The relroviral vector genomes of the present invention for subsequent use in gene therapy preferably contain the minimum retroviral material necessary to function efficiently as vectors. The purpose of this is to allow space for the incorporation of the NOI(s), and for safety reasons. Retroviral vector genomes are preferably replication defective due to the absence of functional genes encoding one or more of the structural (or packaging) components encoded by the gag-pol and env genes. The absent components required for particle production are provided in trans in the producer cell. The absence of virus structural components in the genome also means that undesirable immune responses generated against virus proteins expressed in the target cell are reduced or avoided. Furthermore, possible reconstruction of infectious viral particles is preferably avoided where in vivo use is contemplated. Therefore, the viral structural components are preferably excluded from the genome as far as possible, in order to reduce the chance of any successful recombination.
The retroviral vector particles of the present invention are typically generated in a suitable producer cell. Producer cells are generally mammalian cells but can be for example insect cells. A producer cell may be a packaging cell containing the virus structural genes, normally integrated into its genome. The packaging cell is then transfected with a nucleic acid encoding the vector genome, for the production of infective, replication defective vector particles. Alternatively the producer cell may be co-transfected with nucleic acid sequences encoding the vector genome and the structural components, and/or with the nucleic acid sequences present on one or more expression vectors such as plasmids, adenovirus vectors, herpes viral vectors or any method known to deliver functional DNA into target cells.
In accordance with a highly preferred embodiment of the present invention, we surprisingly discovered that the envelope protein from rabies virus, the rabies G protein, can efficiently pseudotype a wide variety of retroviral vectors. These include not only vectors constructed from murine oncoretroviruses such as MLV, but also vectors constructed from primate lentiviruses such as HIV and from non-primate lentiviruses such as equine infectious anaemia virus (EIAV).
In one embodiment, the vector of the present invention is constructed from or is derivable from a lentivirus. This has the advantage that the vector may be capable of transducing non-dividing cells and dividing cells.
Thus, the preferred retroviral vectors for pseudotyping according to the invention are lentivirus vectors such as HIV or EIAV vectors. These have the advantages noted above. In particular a rabies G pseudotyped lentivirus vector having rabies virus target cell range will be capable of transducing non-dividing cells of the central nervous system such as neurons.
The findings of the present invention are highly surprising. In this respect, although rabies and VSV are Rhabdoviridae, which is a very large family containing five diverse sub-groups, they (i.e. VSV and rabies) are in different sub-groups. Moreover, the rabies G protein has little homology with VSV-G (Rose et al., 1982 J. Virol. 43: 361-364). The rabies G protein also has a much longer cytoplasmic domain than VSV-G, of normally about 44 amino acids (in length) compared with the 28 to 31 amino acid VSV-G cytoplasmic domain. The finding that the rabies G protein is able to pseudotype MLV is therefore unexpected, given that truncation of the 144 amino acid HIV-1 cytoplasmic tail was required for efficient pseudotyping of MLV particles (Mamnmano et al., 1997 J. Virol. 71:3341-3345). It is also surprising that the rabies G protein can additionally pseudotype other retroviruses such as viruses in the lentivirus group.
The invention therefore provides in one aspect a retroviral vector particle pseudotyped with a rabies virus G protein.
In another aspect, the invention provides a retroviral vector production system comprising a nucleic acid sequence which encodes a rabies virus G protein, a nucleic acid sequence which encodes a retrovirus vector genome and optionally one or more further nucleic acid sequences which encode packaging components required for the generation of infective retroviral vector particles containing the genome.
In a further aspect, the invention provides the use of a rabies virus G protein to alter the target cell range of a retroviral vector.
In another aspect, the invention provides a method of producing retroviral vector particles having an envelope comprising rabies virus ( protein, which method comprises providing a retroviral vector production system as described herein, in a producer cell, subjecting the producer cell to conditions suitable for the expression of vector particle components and the production of vector particles, and harvesting the vector particles from the supernatant.
In yet another aspect, the invention provides a method of transducing a target cell with a NOI, which method comprises contacting the cell with a retroviral vector particle as described herein, carrying the NOI, under conditions to allow attachment to and entry of the vector into the cell such that the NOI enters :the target cell genome.
In addition to the rabies G protein present in the envelope of a vector according to the invention, one or more other envelope proteins may also be present. This may include for example a native envelope protein of the retrovirus. The use of a native envelope protein in addition to a pseudotyping protein can be beneficial to the stability and/or function of the envelope. It may even broaden the infectious profile of the vector.
The present invention also provides a pharmaceutical composition for treating an individual by gene therapy, wherein the composition comprises a therapeutically effective amount of a retroviral vector according to the present invention. The pharmaceutical composition may be for human or animal usage. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient.
The composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise asxe2x80x94or in addition toxe2x80x94the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agents that may aid or increase the viral entry into the target site (such as for example a lipid delivery system).
Where appropriate, the pharmaceutical compositions can be administered by any one or more of: inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
Thus, in summation, the present invention relates to a retroviral vector having a heterologous envelope protein, in particular a rabies virus G protein. The present invention also relates to a retroviral vector production system for the production of retroviral vectors having an envelope comprising a rabies virus G protein, as well as to methods of producing the vector and the systems, and to methods involving the use of the vector and the systems.
The present invention will now be described by way of example only, and with reference to the following Figuresxe2x80x94in which: